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Tissue sections offer the opportunity to understand a patient’s condition, to make better prognostic evaluations and to select optimum treatments, as evidenced by the place pathology holds today in clinical practice. Yet, there is a wealth of information locked up in a tissue section that is only partially accessed, due mainly to the limitations of tools and methods. Often tissues are assessed primarily...
To achieve specificity and sensitivity using immunohistochemistry it is necessary to combine the application of validated primary antibodies with optimised pre-treatment, detection and visualisation steps. The influence of these surrounding procedures is reviewed. A practical evaluation of tyramide signal amplification and rolling circle amplification detection methods is provided in which formalin...
As biomarker discovery takes centre-stage, the role of immunohistochemistry within that process is increasing. At the same time, the number of antibodies being produced for “research use” continues to rise and it is important that antibodies to be used as biomarkers are validated for specificity and sensitivity before use. This guideline seeks to provide a stepwise approach for the validation of an...
Advances in the understanding of molecular pathology and thereby the mechanisms that could be amenable to therapeutic manipulation are the reason that pharmaceutical research and development is focused increasingly on measurement of molecular biomarkers in human biological samples. Obtaining direct or indirect access to sufficient samples that are fit for research purposes can be a major challenge...
It is impossible to underplay the importance of fixation in histopathology. Whether the scientist is interested in the extraction of information on lipids, proteins, RNA or DNA, fixation is critical to this extraction. This review aims to give a brief overview of the current “state of play” in fixation and focus on the effect fixation, and particularly the effect of the newer brand of “molecular fixatives”...
Genetic methods of antibody generation offer a highly tuneable tool for the production of target specific reagents suitable for a wide range of applications, including immunohistochemistry. The direct linkage between binder phenotype and genotype enables the rapid identification and manipulation of specific binders into formats suitable for highly specific and sensitive detection of targets from soluble...
Digital pathology and the adoption of image analysis have grown rapidly in the last few years. This is largely due to the implementation of whole slide scanning, advances in software and computer processing capacity and the increasing importance of tissue-based research for biomarker discovery and stratified medicine. This review sets out the key application areas for digital pathology and image analysis,...
In situ hybridisation (ISH) is an established family of closely related methods for the detection and visualisation of specific nucleic acid sequences (DNA, RNA) in tissue sections, cytological preparations and whole organisms. The technique has a history of refinements and applications going back over several decades and is routinely employed in laboratories where visualisation of gene expression...
Post-translational histone modifications are highly correlated with transcriptional activity, but the relative timing of these marks and their dynamic interplay during gene regulation remains controversial. To shed light on this problem and clarify the connections between histone modifications and transcription, we demonstrate how FabLEM (Fab-based Live Endogenous Modification labeling) can be used...
It remains unclear how the 2m of human genomic DNA is organized in each cell. The textbook model has long assumed that the 11-nm-diameter nucleosome fiber (beads-on-a-string), in which DNA is wrapped around core histones, is folded into a 30-nm chromatin fiber. One of the classical models assumes that the 30-nm chromatin fiber is further folded helically to form a larger fiber. Small-angle X-ray scattering...
Crystallographic and NMR approaches have provided a wealth of structural information about protein domains. However, often these domains are found as components of larger multi domain polypeptides or complexes. Orienting domains within such contexts can provide powerful new insight into their function. The combination of site specific spin labelling and Pulsed Electron Double Resonance (PELDOR) provide...
The importance of Chromatin Immunoprecipitation (ChIP) technology has grown exponentially along with an increased interest in epigenetic regulation. The correlation of transcription factors with histone marks is now well established as the center of epigenetic studies; therefore, precise knowledge about histone marks is critical to unravel their molecular function and to understand their role in biological...
Transcription factor binding sites in chromatin are routinely inventoried by the chromatin immunoprecipitation assay, and these binding patterns can provide precise and detailed information about cell state. However, some fundamental molecular questions regarding transcription factor function require an understanding of in vivo binding dynamics as well as location information. Here we describe the...
Nucleosomes are extremely stable histone–DNA complexes that form the building blocks of chromatin, which accommodates genomic DNA within the nucleus. The dynamic properties of chromatin play essential roles in regulating genomic DNA functions, such as DNA replication, recombination, repair, and transcription. Histones are the protein components of nucleosomes, and their diverse modifications and variants...
Histone proteins undergo various types of post-translational modifications (PTMs) to regulate dynamic processes in the cell, including replication, transcription and DNA damage repair. One type of histone PTM is the attachment of a small protein, ubiquitin (Ub). In eukaryotic organisms, a single Ub is attached to specific lysine residues of histones H2A and H2B in a modification that, unlike many...
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